Antigenic variation enables pathogens to avoid the host immune response by continual switching of surface proteins. The protozoan blood parasite Trypanosoma brucei causes human African ...trypanosomiasis ("sleeping sickness") across sub-Saharan Africa and is a model system for antigenic variation, surviving by periodically replacing a monolayer of variant surface glycoproteins (VSG) that covers its cell surface. We compared the genome of Trypanosoma brucei with two closely related parasites Trypanosoma congolense and Trypanosoma vivax, to reveal how the variant antigen repertoire has evolved and how it might affect contemporary antigenic diversity. We reconstruct VSG diversification showing that Trypanosoma congolense uses variant antigens derived from multiple ancestral VSG lineages, whereas in Trypanosoma brucei VSG have recent origins, and ancestral gene lineages have been repeatedly co-opted to novel functions. These historical differences are reflected in fundamental differences between species in the scale and mechanism of recombination. Using phylogenetic incompatibility as a metric for genetic exchange, we show that the frequency of recombination is comparable between Trypanosoma congolense and Trypanosoma brucei but is much lower in Trypanosoma vivax. Furthermore, in showing that the C-terminal domain of Trypanosoma brucei VSG plays a crucial role in facilitating exchange, we reveal substantial species differences in the mechanism of VSG diversification. Our results demonstrate how past VSG evolution indirectly determines the ability of contemporary parasites to generate novel variant antigens through recombination and suggest that the current model for antigenic variation in Trypanosoma brucei is only one means by which these parasites maintain chronic infections.
Full text
Available for:
BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Identification of replication initiation sites, termed origins, is a crucial step in understanding genome transmission in any organism. Transcription of the Trypanosoma brucei genome is highly ...unusual, with each chromosome comprising a few discrete transcription units. To understand how DNA replication occurs in the context of such organization, we have performed genome-wide mapping of the binding sites of the replication initiator ORC1/CDC6 and have identified replication origins, revealing that both localize to the boundaries of the transcription units. A remarkably small number of active origins is seen, whose spacing is greater than in any other eukaryote. We show that replication and transcription in T. brucei have a profound functional overlap, as reducing ORC1/CDC6 levels leads to genome-wide increases in mRNA levels arising from the boundaries of the transcription units. In addition, ORC1/CDC6 loss causes derepression of silent Variant Surface Glycoprotein genes, which are critical for host immune evasion.
Display omitted
► DNA replication origins are widely dispersed in T. brucei chromosome cores ► Origins and ORC1/CDC6 localize at the boundaries of multigene transcription units ► Localization of T. brucei ORC1/CDC6 is distinct in chromosome cores and subtelomeres ► ORC1/CDC6 acts in transcription regulation, including of some VSGs, in T. brucei
Designation of DNA replication origins, the sites where DNA synthesis initiates, remains poorly understood in most eukaryotic genomes, including the relationship of this reaction with transcription. McCulloch, Bell, and colleagues now map origins and the binding sites of a key replication initiator, termed ORC1/CDC6, in the African trypanosome genome, which is highly unusual among eukaryotes in that each chromosome comprises a few discrete multigene transcription units. These data, coupled with global transcript mapping, reveal extensive functional overlap between DNA replication and transcription.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
DNA replication initiates by formation of a pre-replication complex on sequences termed origins. In eukaryotes, the pre-replication complex is composed of the Origin Recognition Complex (ORC), Cdc6 ...and the MCM replicative helicase in conjunction with Cdt1. Eukaryotic ORC is considered to be composed of six subunits, named Orc1-6, and monomeric Cdc6 is closely related in sequence to Orc1. However, ORC has been little explored in protists, and only a single ORC protein, related to both Orc1 and Cdc6, has been shown to act in DNA replication in Trypanosoma brucei. Here we identify three highly diverged putative T. brucei ORC components that interact with ORC1/CDC6 and contribute to cell division. Two of these factors are so diverged that we cannot determine if they are eukaryotic ORC subunit orthologues, or are parasite-specific replication factors. The other we show to be a highly diverged Orc4 orthologue, demonstrating that this is one of the most widely conserved ORC subunits in protists and revealing it to be a key element of eukaryotic ORC architecture. Additionally, we have examined interactions amongst the T. brucei MCM subunits and show that this has the conventional eukaryotic heterohexameric structure, suggesting that divergence in the T. brucei replication machinery is limited to the earliest steps in origin licensing.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Few studies are available evaluating the impact of rapid-acting insulin analogues on long-term diabetes outcomes. Our aim was to compare the use of rapid-acting insulin analogues versus human regular ...insulin in relation to the occurrence of diabetic complications in a cohort of diabetic patients through the analysis of administrative databases.
A population-based cohort study was conducted using administrative data from four local health authorities in the Abruzzo Region (900,000 inhabitants). Diabetic patients free of macrovascular disease at baseline and treated either with human regular insulin or rapid-acting insulin analogues were followed for a maximum of 3 years. The incidence of diabetic complications was ascertained by hospital discharge claims. Hazard ratios (HRs) and 95% CIs of any diabetic complication and macrovascular, microvascular and metabolic complications were estimated separately using Cox proportional hazard models adjusted for patients' characteristics and anti-diabetic drug use. Propensity score matching was also used to adjust for significant difference in the baseline characteristics between the two treatment groups.
A total of 2,286 patients were included: 914 receiving human regular insulin and 1,372 rapid-acting insulin analogues. During the follow-up, 286 (31.3%) incident events occurred in the human regular insulin group and 235 (17.1%) in the rapid-acting insulin analogue group. After propensity score-based matched-pair analyses, rapid-acting insulin analogues users had a HR of 0.73 (0.58-0.92) for any diabetes-related complication and HRs of 0.73 (0.55-0.93) and 0.55 (0.32-0.96) for macrovascular and metabolic complications respectively, as compared with human regular insulin users. No difference between the two groups was found for microvascular complications.
Our findings suggest that the use of rapid-acting insulin analogues is associated with a lower risk of cardiovascular and metabolic complications compared with human regular insulin use.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Rivers support some of Earth's richest biodiversity
and provide essential ecosystem services to society
, but they are often fragmented by barriers to free flow
. In Europe, attempts to quantify ...river connectivity have been hampered by the absence of a harmonized barrier database. Here we show that there are at least 1.2 million instream barriers in 36 European countries (with a mean density of 0.74 barriers per kilometre), 68 per cent of which are structures less than two metres in height that are often overlooked. Standardized walkover surveys along 2,715 kilometres of stream length for 147 rivers indicate that existing records underestimate barrier numbers by about 61 per cent. The highest barrier densities occur in the heavily modified rivers of central Europe and the lowest barrier densities occur in the most remote, sparsely populated alpine areas. Across Europe, the main predictors of barrier density are agricultural pressure, density of river-road crossings, extent of surface water and elevation. Relatively unfragmented rivers are still found in the Balkans, the Baltic states and parts of Scandinavia and southern Europe, but these require urgent protection from proposed dam developments. Our findings could inform the implementation of the EU Biodiversity Strategy, which aims to reconnect 25,000 kilometres of Europe's rivers by 2030, but achieving this will require a paradigm shift in river restoration that recognizes the widespread impacts caused by small barriers.
Full text
Available for:
FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Trypanosoma brucei evades host acquired immunity through differential activation of its large archive of silent variant surface glycoprotein (VSG) genes, most of which are pseudogenes in subtelomeric ...arrays. We have analyzed 940 VSGs, representing one half to two thirds of the arrays. Sequence types A and B of the VSG N-terminal domains were confirmed, while type C was found to be a constituent of type A. Two new C-terminal domain types were found. Nearly all combinations of domain types occurred, with some bias to particular combinations. One-third of encoded N-terminal domains, but only 13% of C-terminal domains, are intact, indicating a particular need for silent VSGs to gain a functional C-terminal domain to be expressed. About 60% of VSGs are unique, the rest occurring in subfamilies of two to four close homologs (>50%-52% peptide identity). We found a subset of VSG-related genes, differing from VSGs in genomic environment and expression patterns, and predict they have distinct function. Almost all (92%) full-length array VSGs have the partially conserved flanks associated with the duplication mechanism that activates silent genes, and these sequences have also contributed to archive evolution, mediating most of the conversions of segments, containing >/=1 VSG, within and between arrays. During infection, intact array genes became activated by duplication after two weeks, and mosaic VSGs assembled from pseudogenes became expressed by week three and predominated by week four. The small subfamily structure of the archive appears to be fundamental in providing the interacting donors for mosaic formation.
Antigenic variation is an immune evasion strategy that has evolved in viral, bacterial and protistan pathogens. In the African trypanosome this involves stochastic switches in the composition of a ...variant surface glycoprotein (VSG) coat, using a massive archive of silent VSG genes to change the identity of the single VSG expressed at a time. VSG switching is driven primarily by recombination reactions that move silent VSGs into specialized expression sites, though transcription-based switching can also occur. Here we discuss what is being revealed about the machinery that underlies these switching mechanisms, including what parallels can be drawn with other pathogens. In addition, we discuss how such switching reactions act in a hierarchy and contribute to pathogen survival in the face of immune attack, including the establishment and maintenance of chronic infections, leading to host-host transmission.
Full text
Available for:
BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
Artificial barriers are one of the main threats to river ecosystems, resulting in habitat fragmentation and loss of connectivity. Yet, the abundance and distribution of most artificial barriers, ...excluding high-head dams, is poorly documented. We provide a comprehensive assessment of the distribution and typology of artificial barriers in Great Britain, and estimate for the first time the extent of river fragmentation. To this end, barrier data were compiled from existing databases and were ground-truthed by field surveys in England, Scotland and Wales to derive a correction factor for barrier density across Great Britain. Field surveys indicate that existing barrier databases underestimate barrier density by 68%, particularly in the case of low-head structures (<1 m) which are often missing from current records. Field-corrected barrier density estimates ranged from 0.48 barriers/km in Scotland to 0.63 barriers/km in Wales, and 0.75 barriers/km in England. Corresponding estimates of stream fragmentation by weirs and dams only, measured as mean barrier-free length, were 12.30 km in Scotland, 6.68 km in Wales and 5.29 km in England, suggesting the extent of river modification differs between regions. Our study indicates that 97% of the river network in Great Britain is fragmented and <1% of the catchments are free of artificial barriers.
Display omitted
•Ground truthed first assessment of stream fragmentation across Great Britain.•Existing barrier databases underestimate stream fragmentation by at least 68%.•There is at least one artificial barrier every 1.5 km of stream in Great Britain.•Only 3.3% of the total river network of Great Britain is fully connected.•Only 1% of the rivers in England, Scotland and Wales are free of artificial barriers.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Trypanosomes are coated with a variant surface glycoprotein (VSG) that is so densely packed that it physically protects underlying proteins from effectors of the host immune system. Periodically ...cells expressing a distinct VSG arise in a population and thereby evade immunity. The main structural feature of VSGs are two long alpha-helices that form a coiled coil, and sets of relatively unstructured loops that are distal to the plasma membrane and contain most or all of the protective epitopes. The primary structure of different VSGs is highly variable, typically displaying only ~20% identity with each other. The genome has nearly 2000 VSG genes, which are located in subtelomeres. Only one VSG gene is expressed at a time, and switching between VSGs primarily involves gene conversion events. The archive of silent VSGs undergoes diversifying evolution rapidly, also involving gene conversion. The VSG family is a paradigm for alpha helical coiled coil structures, epitope variation and GPI-anchor signals. At the DNA level, the genes are a paradigm for diversifying evolutionary processes and for the role of subtelomeres and recombination mechanisms in generation of diversity in multigene families. To enable ready availability of VSG sequences for addressing these general questions, and trypanosome-specific questions, we have created VSGdb, a database of all known sequences.
VSGdb contains fully annotated VSG sequences from the genome sequencing project, with which it shares all identifiers and annotation, and other available sequences. The database can be queried in various ways. Sequence retrieval, in FASTA format, can deliver protein or nucleotide sequence filtered by chromosomes or contigs, gene type (functional, pseudogene, etc.), domain and domain sequence family. Retrieved sequences can be stored as a temporary database for BLAST querying, reports from which include hyperlinks to the genome project database (GeneDB) CDS Info and to individual VSGdb pages for each VSG, containing annotation and sequence data. Queries (text search) with specific annotation terms yield a list of relevant VSGs, displayed as identifiers leading again to individual VSG web pages.
VSGdb http://www.vsgdb.org/ is a freely available, web-based platform enabling easy retrieval, via various filters, of sets of VSGs that will enable detailed analysis of a number of general and trypanosome-specific questions, regarding protein structure potential, epitope variability, sequence evolution and recombination events.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Bacteria are primarily responsible for biological water treatment processes in constructed wetland systems. Gravel in constructed wetlands serves as an essential substrate onto which complex ...bacterial biofilms may successfully grow and evolve. To fully understand the bacterial community in these systems it is crucial to properly isolate biofilms and process DNA from such substrates. This study looked at how best to isolate bacterial biofilms from gravel substrates in terms of bacterial richness. It considered factors including the duration of agitation during extraction, extraction temperature, and enzyme usage. Further, the 16S taxonomy data subsequently produced from Illumina MiSeq reads (using the SILVA 132 ribosomal RNA (rRNA) database on the DADA2 pipeline) were compared with the 16S data produced from Oxford Nanopore Technologies (ONT) MinION reads (using the NCBI 16S database on the EPI2ME pipeline). Finally, performance was tested by comparing the taxonomy data generated from the Illumina MiSeq and ONT MinION reads using the same (SILVA 132) database. We found no significant differences in the effective number of species observed when using different bacterial biofilm detachment techniques. However, enzyme treatment enhanced the total concentration of DNA. In terms of wetland community profiles, relative abundance differences within each sample type were clearer at the genus level. For genus-level taxonomic classification, MinION sequencing with the EPI2ME pipeline (NCBI database) produced bacterial abundance information that was poorly correlated with that from the Illumina MiSeq and DADA2 pipelines (SILVA132 database). When using the same database for each sequencing technology (SILVA132), the correlation between relative abundances at genus-level improved from negligible to moderate. This study provides detailed information of value to researchers working on constructed wetlands regarding efficient biofilm detachment techniques for DNA isolation and 16 s metabarcoding platforms for sequencing and data analysis.
•Different detachment techniques did not change the effective number of species.•Enzyme treatment enhanced the total concentration of DNA.•Wetland samples showed differences in the genus-level bacterial relative abundance.•Genus-level correlation was poor between MiSeq and MinION using different databases.•Genus-level correlation was moderate between MiSeq and MinION using the same database.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
PDF
